Development of a Cell Line Model for ROS Studies Using Silver Nanoparticles in Rheumatoid Arthritis

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by persistent inflammation and oxidative stress, with reactive oxygen species (ROS) playing a key role in its pathogenesis. Silver nanoparticles (AgNPs) possess redox-modulating and immunomodulatory properties and are being explored for therapeutic applications. However, their dose- and cell-specific effects in RA-relevant models remain poorly understood. Aim: To develop an in vitro platform using K562 cells, peripheral blood mononuclear cells (PBMCs), and RAW 264.7 macrophages to investigate the cytotoxic, redox-modulating, and immunomodulatory effects of AgNPs in RA. This model evaluates ROS generation, immune biomarker expression, and cell cycle alterations in immune-relevant cellular systems. Study Design: Experimental in vitro study. Place and Duration of Study: Amplikon Biosystems and Loyola Academy, Hyderabad, Telangana, India; April–June 2025. Methodology: AgNPs were synthesized via trisodium citrate reduction and characterized using UV–Vis spectroscopy. PBMCs were isolated from human blood using Ficoll–Paque density gradient centrifugation. K562 cells, PBMCs, and RAW 264.7 cells were treated with varying AgNP concentrations, while lipopolysaccharide (LPS) served as a pro-oxidant control in RAW 264.7 cells. Cytotoxicity was assessed using the Alamar Blue assay, ROS levels by DCFDA assay, immune biomarkers (BCR-ABL in K562, CD69 in PBMCs) by indirect ELISA, and PBMC cell cycle profiles via flow cytometry. Results: UV–Vis spectroscopy confirmed AgNP formation with a peak at ~420 nm. PBMCs showed high viability post-isolation. K562 cells exhibited minimal cytotoxicity, whereas PBMCs displayed dose-dependent toxicity. AgNPs reduced ROS in K562 cells but increased ROS in RAW 264.7 macrophages. LPS treatment significantly elevated ROS in RAW 264.7 cells. ELISA revealed mild BCR-ABL upregulation in K562 cells and significant CD69 downregulation in PBMCs. Flow cytometry indicated G1-phase arrest in PBMCs, suggesting redox-mediated cell cycle modulation. Conclusion: This study presents a novel multi-cellular in vitro model to assess AgNP-induced oxidative and immunological effects in RA. The findings demonstrate cell-specific ROS modulation, immune biomarker changes, and cell cycle alterations, supporting the potential of AgNPs for therapeutic development in autoimmune disorders.